Publications using the System 3

Noise‐induced hearing loss (NIHL) is a common cause of acquired sensorineural hearing loss. Excessive endoplasmic reticulum (ER) stress‐induced apoptosis of cochlear hair cells contributes to NIHL. ER autophagy (ER‐phagy) is a critical pathway for maintaining ER homeostasis and cell survival. DDRGK1 (DDRGK domain containing 1) is a crucial receptor in ER‐phagy, essential for the removal of injured ER components. This work investigates the involvement of DDRGK1‐mediated ER‐phagy in NIHL, which has remained unclear. ER‐phagy flux is inhibited in HEI‐OC1 cells treated with hydrogen peroxide. Noise exposure reduces XIAP (X‐linked inhibitor of apoptosis protein) and DDRGK1 protein levels in these cells. Moreover, XIAP binds to DDRGK1, increasing the stability of DDRGK1 and activating ER‐phagy. Notably, in noise‐exposed CBA/CaJ mice, gastrodin, a traditional Chinese medicine ingredient, reduces noise‐induced loss of cochlear hair cells, ribbon synaptic damage, and hearing loss by promoting XIAP expression, thereby increasing DDRGK1 protein levels and activating ER‐phagy. These findings highlight XIAP‐DDRGK1‐mediated ER‐phagy as a novel therapeutic target for NIHL treatment.

Many people have trouble understanding speech in noisy environments despite normal audiometric thresholds, a condition referred to as “hidden hearing loss.” A leading hypothesis attributes this deficit to inner hair cell synaptic pathology (cochlear synaptopathy), which can persist after recovery from noise-induced temporary elevation of audiometric thresholds. This pathology impairs the temporally precise sound encoding necessary for spatial hearing, but direct evidence in primates, human or nonhuman, is lacking. Here, we show that a single noise exposure, producing only a temporary threshold elevation, induced long-lasting alterations in synaptic morphology, without synapse loss, resulting in persistent spatial hearing deficits for up to 11 months post-exposure in nonhuman primates of both sexes. These perceptual deficits were accompanied by a reduction in a neurophysiological measure of binaural processing - the binaural interaction component (BIC) of the auditory brainstem response (ABR). Both behavioral and neural deficits persisted despite full recovery of audiometric thresholds. Together, these findings provide the first evidence in primates that noise exposure disrupts spatial hearing and binaural neural circuit function without loss of hair cells or synapses. Because spatial hearing tests and the ABR/BIC are clinically accessible, this work also establishes translational biomarkers for early neural dysfunction underlying hidden hearing loss.

Bilateral cochlear implant (BiCI) users do not perform as well as typical hearing listeners when discriminating the direction of sound motion. This is likely due to the lack of synchronization between two independently operating sound processors. The use of bilaterally linked processors can improve the timing of electrical stimulation across the ears. However, these improvements are potentially counteracted by spectral peak-picking in sound coding strategies that may activate different electrodes across the ears, thereby reducing the fidelity of the binaural cues that BiCI users rely on for sound localization. To improve interaural synchrony, a bilateral peak-picking strategy that guarantees coordinated stimulation across the ears was developed and tested using the CCi-MOBILE, a bilaterally linked research processor. The auditory motion tracking abilities of nine BiCI users were measured using the bilateral peak-picking strategy and compared to performance with commercially available, unsynchronized processors. Results showed no effect of bilateral peak-picking. However, a small benefit was observed when tracking the range of motion of a sound when listening with bilateral synchronization which appears to be due to reduced interaural level difference changes with auditory motion. These findings suggest that dynamic auditory perception with BiCI may likely need bilaterally-synchronized hardware.

Sensorineural hearing loss (SNHL) is significantly more prevalent in individuals with chronic kidney disease (CKD) than in the general population. Although a strong independent association has been observed between kidney dysfunction and the risk of hearing loss, the underlying mechanisms linking these conditions remain poorly understood. This study investigated the pathophysiology of hearing impairment using adriamycin nephropathy (AN), a well-established animal model of CKD. AN was induced in male BALB/c mice using 10 or 12 mg/kg of adriamycin (ADR), resulting in severe kidney injury and concurrent hearing loss. ADR-treated mice exhibited significant glomerular injury, podocyte damage, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL), along with increased serum creatinine and blood urea nitrogen levels. Hearing impairment was evident after 4-8 weeks of ADR treatment, as assessed by auditory brainstem response and distortion-product otoacoustic emissions, and was accompanied by cochlear hair cell loss and ribbon synapse degeneration. AN affected cochlear function by altering ion channel expression in the stria vascularis and inducing blood-labyrinth barrier (BLB) hyperpermeability, along with changes in endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes. AN significantly increased cochlear NGAL and NLRP3 levels at 4 and 8 weeks following ADR administration. NGAL was highly expressed in the tectorial membrane, cochlear neurons, and organ of Corti, while its receptor, 24p3R, was co-localized with NGAL. These findings demonstrated the role of NGAL and BLB disruption in ADR-induced SNHL, providing novel insights into the mechanistic link between CKD and hearing loss.

Although age-related hearing loss (ARHL) is the most common sensory loss in older adults, underlying mechanisms remain unclear. Recent genome-wide association studies (GWAS) linked variation in several genes with ARHL risk, including KLHDC7B , a gene of unknown function not previously linked to hearing. We demonstrate Klhdc7b is expressed exclusively in sensory hair cells in mouse cochlea. Utilizing two independent mouse knockout models ( Klhdc7b IMPC-/- and Klhdc7b Regn Δ/Δ ) we find that absence of Klhdc7b leads to severe early-onset, progressive hearing loss. Hair cells appear to develop normally, but outer hair cells are progressively lost from base-to-apex of the cochlea, a common pathology in ARHL. These results suggest KLHDC7B is required for maintenance rather than development, of auditory function. The validation in mouse of a human ARHL GWAS association suggests other novel candidates should be investigated. Our work provides two mouse models to study KLHDC7B function, and for development of therapeutic tools for ARHL.

Auditory hypersensitivity is a prominent symptom in Fragile X syndrome (FXS), the most prevalent monogenic cause of autism and intellectual disability. FXS arises through the loss of the protein encoded by the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene, FMRP, required for normal neural circuit excitability. In the brainstem, FMRP is necessary for normal development of acoustic reactivity, and its loss has been implicated in audiogenic seizures (AGS) in Fmr1 knockout (KO) mice, modeling auditory hypersensitivity and seizures in FXS patients. The present study investigated the correlation between auditory brainstem function and behavioral expression of AGS at the early (postnatal day P20, infancy) and late (P32, juvenile) stages of auditory development in Fmr1 KO mice compared with wildtype (WT) mice, and in both females and males. We tested responsiveness to pure tones of select auditory pathway elements through auditory brainstem responses, and neural synchronization to amplitude envelopes of modulated acoustic stimuli through auditory steady‐state responses. AGS behavior was categorized for severity during 5‐min exposure to loud sound. Expression of the immediate early gene cFos was quantified as a marker for neuronal activity in the inferior colliculus. During infancy, more severe AGS expression in Fmr1 KO mice compared with WT mice was accompanied by increased responsiveness to acoustic stimuli at the level of the superior olivary complex and inferior colliculus, and stronger neural synchronicity in subcortical auditory neurons. Fmr1 KO mice also had higher cFos positive cell counts in the inferior colliculus after exposure to loud sound. With age, both AGS susceptibility and exaggerated acoustic stimulus‐evoked activity in the Fmr1 KO mice subsided. Intriguingly, Fmr1 KO mice displayed an altered developmental profile in both the threshold and amplitude of auditory brainstem response. Our findings support evidence that AGS activity relies upon hyperexcitability in the auditory system, including in the lower brainstem, possibly due to disturbed auditory maturation. Hyper‐synchronization to modulated sounds in subcortical auditory neurons seemed to predict AGS severity. The developmental trajectory of the auditory hyperresponsiveness and hypersynchrony suggests a transient processing alteration underlying heightened AGS susceptibility in Fmr1 KO mice. A better understanding of FXS‐related circuit and behavioral symptoms of auditory processing across development provides the potential to identify therapeutic strategies to achieve auditory function recovery in FXS.

No pharmacological interventions exist that can restore or preserve auditory function in the mammalian cochlea. Auditory hair cells (HCs) do not spontaneously regenerate, leading to permanent hearing loss. In non-mammalian vertebrates, HC regeneration happens through proliferation and differentiation of their clonally related supporting cells (SCs). The present study supports the potential of quinoxaline (Qx), a nonsteroidal anti-inflammatory compound, to stimulate SC proliferation in the auditory sensory epithelium, a process that may prime the tissue for future HC regeneration. We synthesized a series of Qx derivatives by introducing various substitutions, ranging from hydrophilic to lipophilic. Seventy analogs were generated and tested in vitro and in vivo. Among those, only one (Qx-100) exhibited the best medicinal chemistry profile and was further modified to expand the structure–activity relationship of the chemotype, develop additional analogs, and optimize potency, bioavailability, and in vivo efficacy. Ten new lead variants were generated. Of those, Qx-294 and Qx-301 demonstrated promising in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) profiles and were selected for further testing. Overall, both compounds were rapidly absorbed in zebrafish and mice and promoted cell proliferation in vitro and in vivo without signs of apoptosis, supporting their potential for sensory HC regeneration.

The gene therapy achieved by AAV-mediated otoferlin-overexpression is an effective therapeutic strategy for congenital deafness. However, achieving its physiological and endogenous patterns of expression remains challenging. Here, we generate the homologous mutation Otof c.1315 C > T (p.R439*), equivalent to OTOF c.1273 C > T (p.R425*) found in humans with profound deafness, to create a nonsense mutation-induced deaf mouse model. We then deliver the ‘RESTART v3’ system, which is a CRISPR-free RNA base editor for nonsense mutation suppression, into the cochlea of the mice. We achieve physiological otoferlin expression, and the edited premature termination codon is reverse-mutated to the original amino acid. We observe significant hearing restoration and enhancement of the behavioral auditory startle reflex. Thus, our study presents a successful RNA editing strategy to significantly restore hereditary deafness in mice carrying the specific Otof nonsense mutation, which holds great promise for future clinical translation.

Ciliogenesis requires an orchestrated interaction between the Golgi apparatus and centrioles via vesicle trafficking, yet this process is still poorly understood. Phosphatidylinositol 4-kinase β (PI4KB) is a conserved kinase that localizes to the Golgi for generating phosphatidylinositol 4-phosphate, an important lipid component related to cilium formation. Here, we demonstrate a previously uncharacterized mechanism of PI4KB in regulating Rab11a to enable proper ciliogenesis. PI4KB kinase activity maintains the normal vesicle density around the Golgi and Rab11a localization to centrioles in ciliogenesis. Inhibition of PI4KB activity leads to the reduced centriole localization but accumulation of Rab11a on the Golgi. We identified that the activation of Rab11a relies on PI4KB activity, while inactive Rab11a–guanosine diphosphate stably associates with Golgi and fails to undergo outward delivery. Autosomal-dominant nonsyndromic sensorineural hearing loss mutations in PI4KB abnormally intensify PI4KB interaction with Rab11a, leading to the aberrant subcellular Rab11a localization and defective ciliogenesis. Collectively, our study delineates a critical role for PI4KB in post–Golgi vesicle formation and ciliogenesis.

Damage to inner hair cells (IHCs) is a leading cause of hearing loss, typically initiating at the base region of the basilar membrane. However, the mechanisms and preventative strategies for IHC damage remain to be elucidated. This study revealed that IHCs in the low-frequency region exhibit a significantly faster calcium clearance rate than high-frequency IHCs. This difference is associated with different PMCA1 expression. We then generated an IHC-specific Pmca1 knockout mouse model ( Pmca1 CKO) exhibiting profound hearing loss and IHC death. Using single-cell RNA-seq analysis, we found that the differentially expressed genes (DEGs) were related to tetrahydrofolate biosynthesis, DNA damage, and DNA repair dysfunction. We therefore treated Pmca1 CKO mice with folic acid and found that it protected IHCs by reducing γ-H2A.X levels. In addition, we found that folic acid protected IHCs from noise-induced damage. Overall, our findings suggest that disrupted calcium homeostasis plays a role in IHC damage and that folic acid may be a promising therapeutic agent for protecting hair cells.

Introduction Age-related hearing loss (presbycusis) is the most common sensory deficit in the elderly, yet it lacks effective pharmacological treatments. The decline of the cyclic AMP (cAMP) signaling pathway is implicated in aging, but its functional role in the auditory system remains largely unknown. Methods To investigate whether enhancing cAMP signaling could counteract presbycusis and elucidate the underlying molecular mechanism, we used a D-galactose-induced cellular senescence model and an in vivo aged mouse model. The effects of the cAMP analog dibutyryl-cAMP (dbcAMP) on cellular senescence, inflammation, mitochondrial function, and cochlear structure were evaluated. The role of Mitofusin-2 (MFN2) was further assessed using siRNA-mediated knockdown. Results dbcAMP potently suppressed cellular senescence and the associated inflammatory phenotype. In addition, cAMP treatment alleviated mitochondrial dysfunction, as indicated by improved mitochondrial bioenergetics and morphology. The anti-senescence effects of cAMP were significantly blunted upon siRNA-mediated knockdown of Mfn2, establishing MFN2 as a key, though perhaps not the sole, component of this protective pathway. Importantly, systemic administration of dbcAMP to aged mice significantly preserved hearing function and protected cochlear hair cells. This in vivo protection was accompanied by an upregulation of cochlear MFN2 and a coordinated suppression of senescence and inflammation markers. Discussion Our study reveals a critical protective role for the cAMP-MFN2 axis in auditory aging by suppressing cellular senescence. These findings identify this axis as a novel and promising therapeutic target for the treatment of age-related hearing loss.

Objectives: Following deafness, a cochlear implant (CI) can be used for the restoration of hearing. CI effectiveness relies on the condition of the auditory nerve, which typically degenerates after deafness. The nerve’s condition can be assessed with the electrically evoked compound action potential (eCAP), the whole-nerve response to an electric pulse. Changes in the eCAP following an increase in the inter-phase gap (IPG) of a biphasic pulse have been reported to be informative of neural survival. This IPG effect can be explained by the temporal separation of the hyperpolarizing phase from the depolarizing phase. We hypothesize that increasing the phase duration (PD) has a similar effect. Design: We investigated the PD effect in normal hearing and in ototoxically deafened guinea pigs (total N = 40) with various conditions of the auditory nerve by recording eCAPs to biphasic current pulses with alternating polarity and with varying PD and IPG. The eCAP data were obtained from both chronically and acutely implanted guinea pigs by using CI stimulation paradigms with a fixed charge and varying PD (30, 50, or 100 µs) and IPG (2.1 or 30 µs). We evaluated six eCAP measures: five derived from the amplitude growth function and the N 1 latency. We examined the relationships of PD and IPG effects with the survival of the spiral ganglion cells. Results: The PD effects were stronger for latency than IPG effects, but weaker for the other five evaluated eCAP measures. The PD effect did not correlate as well with neural survival as the IPG effect. The IPG effect decreased with increasing PD, and accordingly, the stronger correlations between the IPG effect and neural survival were found for a short PD. Notably, the latency increase with increasing PD was greater than 1, which indicates the second phase of the pulse significantly contributes to the eCAP. This second-phase contribution was larger for lower neural survival. Conclusions: The PD effect of latency has predictive power in assessing neural survival. When using other eCAP measures than latency, the best approach to assess neural survival is using the IPG effect with a short PD (around 30 µs).

Background Bmi1, a key component of the Polycomb repressive complex 1, plays a critical role in regulating gene expression by modulating chromatin structure. Its depletion is known to cause hair cell loss in the neonatal mouse cochlea. This study aimed to investigate the epigenetic mechanisms and transcriptional consequences of Bmi1 depletion in the neonatal auditory sensory epithelium. Results Analysis of neonatal Bmi1 knockout mice using H3K27me3 chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing revealed significant transcriptional alterations, particularly in genes governing cell proliferation, senescence, and death. Bmi1 depletion resulted in widespread gene upregulation and increased chromatin accessibility, which correlated with reduced H3K27me3 enrichment. Notably, expression of Cdkn2c , a key cell cycle regulator, was significantly upregulated. Inhibition of Cdkn2c rescued the proliferative capacity of inner ear epithelial cells in Bmi1 knockout mice. Conclusions These findings demonstrate that Bmi1 maintains transcriptional repression and chromatin state in the developing cochlea, primarily through H3K27me3 deposition. Depletion disrupts this control, leading to Cdkn2c overexpression and impaired cell proliferation. This identifies Cdkn2c and its regulatory pathway as potential therapeutic targets for hearing loss associated with hair cell depletion.

Neural circuits undergo experience-dependent plasticity to form long-lasting memories, but how inhibitory interneurons contribute to this process remains poorly understood. Using miniature microscope calcium imaging, we monitored the activity of large amygdala interneuron populations in freely moving mice during fear learning and extinction. Here we show that interneurons exhibit complex and heterogeneous plasticity at both single-cell and ensemble levels across memory acquisition, expression, and extinction. Analysis of molecular interneuron subpopulations revealed that disinhibitory vasoactive intestinal peptide (VIP)-expressing cells are predominantly activated by salient external stimuli, whereas the activity of projection neuron targeting somatostatin (SST) interneurons additionally aligns with internal behavioural states. Although responses within each interneuron subtype are non-uniform, molecular identity biases their functional role, producing weighted circuit outputs that can flexibly regulate excitatory projection neuron activity and plasticity. These findings demonstrate that inhibitory interneurons actively shape the encoding and stability of emotional memories, underscoring their importance in adaptive learning.

Sensory issues are common in autism spectrum disorders (ASD) and can significantly affect daily living. The phenomena of gating and habituation of event-related potentials (ERPs) to repetitive stimuli have been suggested as potential biomarkers reflecting atypical sensory processing in ASD. Sensory hypersensitivity and anxiety are closely related in ASD, and habituation to emotionally evocative stimuli may serve as a more sensitive biomarker for sensory hypersensitivity symptoms. However, previous studies have primarily used tonal stimuli, and there has been little investigation into whether habituation to emotionally evocative sounds is impaired in ASD patients. In this study, we compared the degree of habituation of the P1-N1 peak-to-peak amplitude in response to repeated tones and fearful vocalizations between control and ASD groups. Contrary to expectations, no significant difference was observed for fearful vocalizations between the groups, while ASD patients showed significantly reduced habituation to tonal sounds in the left parieto-occipital region. Furthermore, we found a significant correlation between the degree of habituation to tonal sounds in the left parieto-occipital region and sensory hypersensitivity symptoms in ASD patients, and similar abnormalities in BTBR mice, an animal model of ASD. These results suggest that habituation to tonal sounds, rather than emotionally evocative stimuli, may serve as a translational biomarker reflecting sensory hypersensitivity symptoms.

Speech perception relies on two fundamental acoustic components: spectral and temporal. While spectral information is known to be represented in the auditory cortex through tonotopy, how temporal features are organized has remained unclear. Here, by varying onset rise-ramp steepness and frequencies, we reveal that temporal envelope steepness-a critical cue for phoneme discrimination and sound source perception-is systematically mapped in the mouse auditory cortex. Using widefield calcium imaging, we discovered that the envelope steepness is represented orthogonally to the tonotopic axis, forming a two-dimensional cortical map that mirrors the dual structure of sounds. This organization was observed in primary-like areas but not in higher-order-like areas, indicating distinct auditory processing streams. These findings uncover a principle of cortical organization, suggesting that the auditory cortex encodes sound along two independent axes and thereby provides a neural basis for the parallel processing of complex sounds such as speech and natural acoustic environments.

Sensorineural hearing loss (SNHL) is caused mainly by irreversible damage to sensory hair cells after noise exposure, ototoxic medication, and ageing, with men being more prone to developing SNHL than women are. For animal models, sex-related susceptibility to develop SNHL needs to be taken into account. Ototoxic trauma can be modeled in mice via the systemic administration of furosemide and kanamycin. Several other deafening models have shown differences in response to ototoxic medication between female and male mice. Differences in response to kanamycin-induced ototoxicity in male and female mice have not been studied. Here, we examined sex differences in susceptibility to kanamycin plus furosemide-induced ototoxicity in adult C57BL/6 J mice. Adult (postnatal day 40) female and male C57BL/6 J and Lgr5GFP (C57BL/6 J background) mice were used. The animals were deafened with a single dose of furosemide (100 mg/kg, i.v.) in combination with kanamycin (700 or 900 mg/kg, s.c.). Before deafening, seven and twenty-eight days after deafening, auditory brainstem responses (ABRs) to click stimuli were recorded to evaluate hearing performance. The cochleae were harvested seven or twenty-eight days after the induction of ototoxicity and processed for histology to evaluate hair cell loss. Male mice presented large ABR threshold shifts after ototoxic treatment with 700 mg/kg kanamycin (50 dB median hearing loss). In males, 9/12 mice had threshold shifts greater than 40 dB. However, female mice presented significantly less hearing loss in response to 700 mg/kg kanamycin, as observed via ABRs after ototoxic treatment (32 dB median hearing loss), and only 8/22 female mice presented threshold shifts greater than 40 dB. When treated with 900 mg/kg kanamycin, female mice presented large ABR threshold shifts after ototoxic treatment (50 dB median), and 11/16 mice presented threshold shifts greater than 40 dB. Female C57BL/6 J mice are less susceptible to kanamycin-induced hearing loss than males are and hence need higher doses of kanamycin to reach the same level of hearing loss. This finding is in line with the prevalence of disabling hearing loss in humans, which is 7.3% in males and 4.8% in females, where estrogens have been linked to increased hearing performance and protection against hearing loss.

Introduction Bilateral cochlear implants (BiCIs) do not restore sound localization abilities to the full extent exhibited by typical hearing (TH) listeners, partly due to poor encoding of interaural time differences (ITDs). ITD cues have been provided and investigated using synchronized research processors that ensure the precise delivery of ITD cues. These studies have been conducted in a direct stimulation setting, which bypasses the processor microphones and, in most cases, removes interaural level difference cues (ILDs). To our knowledge, this is the first study that evaluated the efficacy of synchronized stimulation in restoring sensitivity to ITDs in a free field localization experiment. This was made possible by the CCi-MOBILE, a portable and real-time processing research platform that allows for synchronizing microphone inputs. Methods Fourteen BiCI listeners were tested with experimental real-time coding strategies in comparison to unsynchronized clinical processors. We calculated the binaural cues from the acoustic stimuli at the level of microphone input. Results The recordings show that the experimental coding strategies in this study deliver ITDs with greater precision than the clinical strategy. However, psychophysical testing did not show the benefit of an ITD-encoding strategy in improving localization in a free field. The ITD encoding strategies preserved ITDs, which better differentiated unique loudspeaker locations than interaural level differences (ILDs), suggesting that listeners could achieve improved performance if they accessed these cues. As expected, ILDs were similar across all strategies, including the ITD encoding strategies. The lack of improvement in localization performance is likely because ILDs remained to be the dominant cue in acute localization testing, even when ITD cues were available. Discussion Providing BiCI listeners with adequate experiences with ITD cues may be necessary to shift their reliance from ILD dominance to a combined reliance on ILD and ITD cues in free-field conditions. The CCi-MOBILE could enable take-home practice with novel stimulation strategies for extended experiences and long-term evaluation in real-world listening environments.

Hearing relies upon speedy synaptic transmission of sound information from inner hair cells (IHCs) to spiral ganglion neurons. To accomplish this, IHCs use a sophisticated presynaptic machinery including the multi-C 2 domain protein otoferlin that is affected by human deafness mutations. Otoferlin is essential for IHC exocytosis, but how it binds Ca 2+ and the target membrane to serve synaptic vesicle (SV) tethering, docking, and fusion remained unclear. Here, we obtained cryo–electron microscopy structures of otoferlin and employed molecular dynamics simulations of membrane binding. We show that membrane binding by otoferlin involves C 2 B-C 2 G domains and repositions C 2 F and C 2 G domains. Disruption of Ca 2+ -binding sites of the C 2 D domain in mice altered synaptic sound encoding and eliminated the Ca 2+ cooperativity of IHC exocytosis, indicating that it requires the binding of several Ca 2+ -ions by otoferlin. Together, our findings elucidate molecular mechanisms underlying otoferlin-mediated SV docking and support the role of otoferlin as Ca 2+ sensor of SV fusion in IHCs.

We investigated the auditory responses of anesthetized rhesus macaques to complex natural sounds using functional magnetic resonance imaging (fMRI) and a near-awake anesthesia protocol combining low-dose sevoflurane and dexmedetomidine. Eleven animals were scanned while listening to macaque vocalizations and non-vocal sounds. Robust activation was observed in primary and belt auditory cortices as well as subcortical structures, indicating preserved auditory responsiveness under anesthesia. However, unlike in awake animals, selective responses to vocalizations in anterior temporal voice areas (aTVAs) were absent. Instead, vocalization sensitivity emerged in the ventral premotor cortex, prefrontal cortex, and posterior middle temporal gyrus, regions associated with the dorsal auditory stream and multisensory integration. These findings suggest that while anesthesia preserves basic auditory processing, it modulates higher-order cortical responses involved in voice perception. Anesthetized fMRI thus offers a valuable tool for large-scale studies of primary auditory functions, while it must be approached with caution when studying higher-order auditory functions.